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Calcium Flux Assays using the LabChip 3000

ty of the assay can be assessed by examining the cell count rate and the cell viability (e.g. EC50) over time.

CHO-m1 cells are loaded in the chip wells at a density of between 3-5 x 106 cells/mL. The cell count over time is monitored to ensure that sufficient statistics are gathered on the last plate of the run. Figure 5 shows that cell counts are > 100 cells per well after 5 hr. Figure 6 shows the stability of the cell response over a typical run time.

8. Optimize Throughput and Cell Usage
Optimization of throughput is achieved by gathering data on a sufficient number of cells for each sample well visited on all assay plates. Figure 7 shows the dependence of data quality (Z') on cell count per sample. Typically more than 50 cells per sample are required for sufficient statistics to develop high Z' assays.

As cell count numbers are dependent on sample sip time, throughput and data quality can be optimized as needed. In this assay, sample sip times of 12 and 5 s were chosen as low- and high-throughput versions of both agonist and antagonist calcium flux assays. The sip time of degassed buffer between samples should also be chosen to maximize throughput while still maintaining a low dissolved gas content on the chip. Buffer sip times of 5 and 3 s were chosen for 12 and 5 s sample sip times, respectively.

The number of wells sampled during an assay run will affect cell usage. Higher-throughput assays will have lower Z' but reduced cell consumption on a per-well basis. Table 2 summarizes throughput and cell usage for CHO-m1 agonist and antagonist assays.

9. Confirm Z': % Activity
Stability vs. Time over URT. Once assay conditions had been set, the assay was validated. Typical validation criteria include data quality (Z') assessment and assay stabil
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